Introduction 13 13 2  1992 2001 1956 1 14 Fig. 1 1956  13 4 8 13 2 Materials and methods Chemicals 13 2 2 Plant material Spinacia oleracea Growth of plant material; chloroplast isolation; chlorophyll assay; measurement of chloroplast intactness; polarographic measurement of chloroplast activity in suspension The above methods have been described in detail in the accompanying paper (Williams and MacLeod preceding paper). Measurement of chloroplast activity on filter membranes 1981 1987 2 2 −1 −1 2 2 2 −2  −1 2 2 Experiments in the leaf-disc oxygen electrode 2 13 2 2 13 2 13 2 2 13 2 2 Reactions were terminated by dissembling the apparatus as quickly as possible and plunging the chloroplast-containing membrane into a beaker of liquid nitrogen. It was estimated that this procedure took no more than 2.5 s. The beaker containing the membrane was then stored in a freezer at −20°C where the liquid nitrogen was allowed to evaporate. After overnight storage at −20°C the enzymes were denatured and the metabolites were extracted by removing the beaker containing the membranes from the freezer and immediately adding boiling 80% ethanol (75 ml) and boiling for a further 5 min. The membranes were then removed from the extract and thoroughly rinsed with water, adding the washings to the extract. After cooling, the pooled extracts were then evaporated to dryness at 37°C under a stream of dry nitrogen. Design of a specific photosynthesis apparatus Because of the limitations of the oxygen electrode for the type of experiments that were required, it was decided to design and construct an apparatus better suited for the purpose. 2 13 2 12 2 13 2 12 2 2 13 2 13 2 2 13 2 1987 1980 1979 Experiments in the photosynthesis apparatus 2 3  −1 13 2 2 13 2 2 13 2 2 2 Preparation of samples for GC/MS analysis The dried samples from the photosynthesis experiments described above were redissolved in water (2 ml) and then processed for GC/MS analysis according to the following methods. 4 3 1992 3 − + 2 5 Derivatization of dephosphorylated sugar phosphates 1963 GC/MS analysis of derivatized sugars −1 −1 m/z −1 13 13 1992 2001 Identification of sugar phosphates in chloroplast extracts 1992 2001 Quantification by GC/MS of sugar phosphates in chloroplast extracts Quantification of analytes is best achieved by the inclusion in the sample mixture of a known amount of an internal standard having similar chemical and physical characteristics to those of the analytes. 13 13 2 m z 3 3 3 2 + 13 m/z m/z Sugars which were available in the free form with a high degree of purity were dried for 3 days over phosphorous pentoxide under a low vacuum. Approximately 0.02 g of the dried powder or syrup (ketopentoses) was weighed to five decimal places to prepare standard solutions of each sugar. Appropriate dilutions of these were taken to prepare a set of 0.5 mM standard solutions. glycero ido glycero altro 1974 1979 Recovery from the dephosphorylation step was assessed by carrying out parallel dephosphorylations of glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate, all of which were assayed enzymically as the free sugars. The recovery of the monophosphates was 88% and of the bisphosphate was 78%. These figures provided a useful estimate of the likely recoveries of those sugars (above), which could not be assayed in the non-phosphorylated form. glycero ido glycero altro 2 5 m z syn anti m z m z 1 Table 1 a Sugar Intercept Slope r Erythrose −0.017 1.068 0.999 Ribose/Arabinose 0.001 0.625 0.998 Xylulose/Ribulose −0.010 1.077 0.999 Fructose −0.008 0.305 0.995 Glucose −0.011 0.524 0.998 Sedoheptulose −0.013 0.678 0.998 g a- −0.009 0.318 0.996 g i- −0.005 0.177 0.992 a Results and discussion 12 2  13 2  13 2 13 13 2  13 2  2 . 13 2 13 2  13 13 2 2 2 2001 Table 2 a Sugar m z Carbons 13 0 13 1 13 2 13 3 13 4 13 5 Xylose 160 C1-2 67.2 24.2 8.6    205 C4-5 80.3 19.7     262 C1-3 51.0 26.8 13.4 8.9   307 C3-5 61.2 21.5 12.5 4.7   452 C1-5 47.1 21.7 17.9 13.3   b 160 C1-2 66.7 24.8 8.5    262 C1-3 55.5 30.7 13.7    307 C3-5 68.5 18.9 12.6    Xylulose d C4-5 90.3 5.6 4.1    263 C1-3 54.6 26.5 13.5 5.4   364 C1-4 50.0 23.8 15.5 8.1 2.5  452 C1-5 46.5 22.1 16.9 9.9 3.9 0.8 c 160 C1-2 67.0 24.7 8.3    d C4-5 81.9 11.7 6.4    e C1-3 59.1 23.1 12.7 5.1   307 C3-5 66.1 17.6 11.0 5.3   319 C2-5 50.4 24.1 14.7 7.9 2.8  Fructose 205 C5-6 89.5 10.5     263 C1-3 65.9 17.8 11.6 4.8   307 C4-6 68.2 16.9 10.1 4.8   319 C3-6 58.6 20.3 11.8 6.4 2.9  364 C1-4 49.6 22.3 16.2 8.7 3.2  Glucose 160 C1-2 73.6 19.2 7.2    d C5-6 78.0 14.6 7.4    f C4-6 60.9 21.5 11.6 6.1   319 C3-6 36.5 23.1 22.2 12.9 5.3  364 C1-4 33.2 27.3 22.2 12.5 4.9  Unidentified heptulose 307 C5-7 57.4 22.6 12.9 7.1   319 C4-7 26.3 27.0 25.6 15.0 6.1  421 C3-7 24.3 24.5 24.1 16.3 8.0 2.8 Sedoheptulose d C6-7 72.4 19.4 8.3    262 C1-3 41.4 29.6 17.3 9.0 2.6  f C5-7 52.0 25.1 15.0 7.9   319 C4-7 25.5 26.0 25.6 16.2 6.7  364 C1-4 31.7 27.4 22.5 13.2 5.3  466 C1-5 17.0 21.5 26.2 20.0 10.7 4.5 Octulose d C7-8 78.6 14.1 7.4    f C6-8 58.2 22.9 12.7 6.3   319 C5-8 41.8 25.2 19.2 10.2 3.6  331 C4-8 31.2 21.5 21.3 14.5 7.8 3.8 421 C4-8 33.6 21.5 21.0 14.4 6.9 2.6 466 C1-5 34.6 22.3 21.5 13.3 6.0 2.3 a 2 13 15 18 29, 30 syn anti syn anti b c 2001 d m/z e m/z m/z f m z m z 13 2  2 13 m/z 14 14 3 1977 Table 3 − 1 Equipment for experiment Apparatus Leaf disc oxygen electrode 13 2 0 30 0 45 0 30 45 Compound a a b No 1 No 2 No 1 No 2 c 0.00 0.00 0.00 0.00 0.28 0.14 0.55 0.79  0.35 Xylose 0.00 0.39 0.00 0.00 0.00 0.00 0.86 1.10 0.61 Lyxose/Arabinose 0.00 0.63 0.00 0.00 0.00 0.00 1.05 0.68 0.81 Ribose/Ribulose/Xylulose 0.20 5.74 0.21 1.86 4.70 2.54 9.79 14.87 6.61 Fructose 0.29 33.71 0.31 2.67 29.66 23.48 42.06 117.6 58.43 Glucose 0.33 10.55 0.35 6.71 8.21 3.60 15.63 39.38 16.18 Unidentified heptulose 0.00 0.00 0.00 0.00 0.29 0.00 0.85 1.04 0.84 Sedoheptulose 0.01 9.04 0.01 1.06 12.97 1.17 21.95 38.90 16.26 g a 0.000 0.17 0.00 0.28 0.40 0.00 1.53 3.53 d a b 13 2 c d m/z 3  3 13 3 13 2 g- a syn- anti- g- -i- 2 2 2001 m/z m/z 13 m/z m/z g a −1 3 13 4 2 1 g a 13 g a 13 2 1989 Table 4 13 13 2 a Carbon Xylose Lyxose Xylulose Ribose Fructose Glucose Sedoheptulose Unk. Heptulose Octulose C-1 { 24.2, { 24.8, – 12.1 – { 19.2, – – – C-2 8.6 8.5 – 23.8 – 7.2 – – – C-3 b 16.7 – d 14.1 c – 7.6 – C-4 { d – 8.4 { d b 27.5 b,c 54.2 25.0 C-5 0 – 7.1 0 { 10.5, { d b,c,d – c C-6     0 0 { d – d C-7       0  { d C-8         0 a b c m/z m/z d m/z m/z 3 1 2 3 13 1980 13 13 2  1989 13 2 14 2 1983 1 2 2 Fig. 2 vide infra).